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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
17 May 2001 (17.05.2001)
PCT
(10) International Publication Number
WO 01/35071 A2
(51) International Patent Classification 7 ;
G01N 15/14
(21) International Application Number: PCI7US00/3O815
(22) International Filing Date:
10 November 2000 (10. 1 1 .2000)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
60/164,643
10 November 1999 (10.11.1999) US
(71) Applicant (for all designated States except US): MASS-
ACHUSETTS INSTITUTE OF TECHNOLOGY
[US/US]; 77 Massachusetts Avenue, Cambridge, MA
02131 (US).
(72) Inventors; and
(75) Inventors/Applicants (for US only): BRAFF, Rebecca
[US/US]; 488 Columbus Avenue, #4, Boston, MA 02118
(US). VOLDMAN, Joel [US/US]; 35 Davis Square,
#6, Somerville, MA 02144 (US). GRAY, Martha, L.
[US/US]; 226 Pleasant Street, Arlington, MA 02476
(US). SCHMIDT, Martin, A. [US/US]; 78 Ashley Place,
Reading, MA 01867 (US). TONER, Mehmet [US/US];
100 Pilgrim Road, WeUesley, MA 02481 (US).
(74) Agents: ENGELLENNER, Thomas, J. et al.; Nutter, Mc-
Clennen & Fish, LLP, One International Place, Boston, MA
02110-2699 (US).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE,
DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU,
ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS,
LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO,
NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR,
TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
Published:
— Without international search report and to be republished
upon receipt of that report
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
(54) Title: CELL ANALYSIS AND SORTING APPARATUS FOR MANIPULATION OF CELLS
(57) Abstract: A cell analysis and sorting apparatus is capable of monitoring over time the behaviour of each cell in a large popu-
lation of cells. The cell analysis and sorting apparatus contains individually addressable cell locations. Each location is capable of
capturing and holding a single cell, and selectively releasing that cell from that particular location. In one aspect of the invention, the
cells are captured and held in wells, and released using vapor bubbles as a means of cell actuation. In another aspect of the invention,
the cells are captured, held and released using electric fields traps.
WO 01/35071
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CELL ANALYSIS AND SORTING APPARATUS
FOR MANIPULATION OF CELLS
FIELD OF THE INVENTION
This invention relates to cell analysis and sorting devices and methods for
manipulating cells using these devices. More particularly, the invention relates to a cell
analysis and sorting apparatus that can capture and hold single cells at known locations
and then selectively release certain of these cells. A method of manipulating the cells
using the cell analysis and sorting apparatus is also provided.
BACKGROUND OF THE INVENTION
Many recent technological advances have enhanced the study of cellular biology
and biomechanical engineering, most notably by improving methods and devices for
carrying out cellular analysis. For example, in the past decade an explosion in the
number of optical probes available for cell analysis has enabled an increase in the amount
of information gleaned from microscopic and flow cytometric assays. Microscopic
assays allow the researcher to monitor the time-response of a limited number of cells
using optical probes. Flow cytometry, on the other hand, uses optical probes for assays
on statistically significant quantities of cells for sorting into subpopulations.
However, these mechanisms alone are insufficient for time-dependent analysis.
Microscopic assays can only track a few cells over time, and do not allow the user to
track the location of individual cells. With flow cytometry, the user can only observe
each cell once, and can only easily sort a cell population into three subpopulations. Flow
cytometry techniques fail to provide for analysis of the same cell multiple times, or for
arbitrary sorting of subpopulations. These kinds of bulk assay techniques produce mean
statistics, but cannot provide the researcher with distribution statistics.
Advances in microsystems technology have also influenced many applications in
the fields of cell biology and biomedical engineering. Scaling down to the micron level
allows the use of smaller sample sizes than those used in conventional techniques.
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Additionally, the smaller size and ability to make large arrays of devices enables multiple
processes to be run in parallel.
Integrated circuits have been fabricated on silicon chips since the 1950s, and as
processing techniques improve, the size of transistors continues to shrink. The ability to
produce large numbers of complex devices on a single chip sparked interest in fabricating
mechanical structures on silicon as well. The range of applications for micro
electromechanical systems (MEMS) is enormous. Accelerometers, pressure sensors, and
actuators are just a few of the many MEMS devices currently produced. Another
application of MEMS is in biology and medicine. Micromachined devices have been
made for use in drug-delivery, DNA analysis, diagnostics, and detection of cell
properties.
Manipulation of cells is another application of MEMS. For example, in the early
1990 , s, Sato et al. described in his paper, which is hereby incorporated by reference,
Individual and Mass Operation of Biological Cells using Micromechanical Silicon
Devices, Sensors and Actuators, 1990, A21-A23:948-953, the use of pressure differentials
to hold cells. Sato et al. microfabricated hydraulic capture chambers that were used to
capture plant cells for use in cell fusion experiments. Pressure differentials were applied
so that single cells were sucked down to plug an array of holes. Cells could not be
individually released from the array, however, because the pressure differential was
applied over the whole array, not to individual holes.
Bousse et al. in his paper, which is hereby incorporated by reference,
Micromachined Multichannel Systems for the Measurement of Cellular Metabolism,
Sensors and Actuators B, 1994, 20:145-150, described arrays of wells etched into silicon
to passively capture cells by gravitational settling. Multiple cells were allowed to settle
into each of an array of wells where they were held against flow due to the
hydrodynamics resulting from the geometry of the wells. Changes in the pH of the
medium surrounding the cells were monitored by sensors in the bottom of the wells, but
the wells lacked a cell-release mechanism, and multiple cells were trapped in each well.
Another known method of cell capture is dielectrophoresis (DEP). DEP refers to the
action of neutral particles in non-uniform electric fields. Neutral polarizable particles
experience a force in non-uniform electric fields which propels them toward the electric
field maxima or minima, depending on whether the particle is more or less polarizable
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than the medium it is in. By arranging the electrodes properly, an electric field may be
produced to stably trap dielectric particles.
Microfabrication has been utilized to make electrode arrays for cell manipulation
since the late 1980s. Researchers have successfully trapped many different cell types,
including mammalian cells, yeast cells, plant cells, and polymeric particles. Much work
involves manipulating cells by exploiting differences in the dielectric properties of
varying cell types to evoke separations, such as separation of viable from non-viable
yeast, and enrichment of CD34+ stem cells from bone marrow and peripheral blood stem
cells. More relevant work on trapping cells in various two- and three-dimensional
microfabricated electrode geometries has been shown by several groups. However,
trapping arrays of cells with the intention of releasing selected subpopulations of cells has
not yet been widely explored. Additionally, DEP can potentially induce large
temperature changes, causing not only convection effects but also profoundly affecting
cell physiology.
These studies demonstrate that it is possible to trap individual and small numbers
of cells in an array on a chip, but without the ability to subsequently manipulate and
selectively release individual cells. This inability to select or sort based on a biochemical
measurement poses a limitation to the kinds of scientific inquiring that may be of interest.
The currently available mechanisms for carrying out cell analysis and sorting are
thus limited in their applications. There is thus a need for an improved method and
apparatus for sorting and releasing large quantities of cells that can easily and efficiently
be used. In addition, there is a need for an analysis and sorting device that allows the user
to look at each cell multiple times, and to track many cells over time. Finally, there is a
need for a cell sorter that lets the user know the cell locations, and to be able to hold and
selectively release the cells so that the user can arbitrarily sort based on any aspect of the
cells' characteristic during time-responsive assays.
SUMMARY OF THE INVENTION
The present invention provides a cell sorting apparatus that is capable of
monitoring over time the behavior of each cell in a large population of cells. The cell
analysis and sorting apparatus contains individually addressable cell locations. Each
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location is capable of capturing and holding a single cell, and selectively releasing that
cell from that particular location. In one aspect of the invention, the cells are captured
and held in wells, and released using vapor bubbles as a means of cell election. In
another aspect of the invention, the cells are captured, held and released using electric
field traps.
According to one aspect of the present invention, the cell analysis and sorting
apparatus has an array of geometric sites for capturing cells traveling along a fluid flow.
The geometric sites are arranged in a defined pattern across a substrate such that
individual sites are known and identifiable. Each geometric site is configured and
dimensioned to hold a single cell. Additionally, each site contains a release mechanism
to selectively release the single cell from that site. Because each site is able to hold only
one cell, and each site has a unique address, the apparatus allows the user to know the
location of any particular cell that has been captured. Further, each site is independently
controllable so that the user is able to arbitrarily capture cells at select locations, and to
release cells at various locations across the array.
In one embodiment of the present invention, the geometric sites are configured as
wells. As a fluid of cells is flown across the array of specifically sized wells, cells will
fall into the wells and become trapped. Each well is sized and shaped to capture only a
single cell, and is configured such that the cell will not escape into the laminar flow of the
fluid above the well. The single cell can be held inside the well by gravitational forces.
Each well can further be attached via a narrow channel to a chamber located below the
well. Within the chamber is a heating element that is able to induce bubble nucleation,
the mechanism for releasing the cell from the site. The bubble creates volume expansion
inside the chamber which, when filled with fluid, will displace a jet of fluid out of the
narrow channel and eject the cell out of the well. Fluid flow above the well will sweep
the ejected cell away to be either collected or discarded.
In another embodiment of the present invention, the geometric sites are formed
from a three-dimensional electric field trap. Each trap comprises four electrodes arranged
in a trapezoidal configuration, where each electrode represents a corner of the trapezoid.
The electric fields of the electrodes create a potential energy well for capturing a single
cell within the center of the trap. By removing the potential energy well of the trap, the
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cell is ejected out of the site and into the fluid flow around the trap. Ejected cells can
then be washed out and collected or discarded.
In yet another embodiment of the present invention, an integrated system is
proposed. The system can be a microfabrication-based dynamic array cytometcr (p.DAC)
having as one of its components the cell analysis and sorting apparatus previously
described. To analyze a population of cells, the cells can be placed on a cell array chip
containing a plurality of cell sites. The cells are held in place within the plurality of cell
sites in a manner similar to that described above and analyzed, for example, by
photometric assay. Using an optical system to detect fluorescence, the response of the
cells can be measured, with the intensity of the fluorescence reflecting the intensity of the
cellular response. Once the experiment is complete, the cells exhibiting the desired
response, or intensity, may be selectively released into a cell sorter to be further studied
or otherwise selectively processed. Such an integrated system would allow researchers to
also look at the cell's time response.
Further features and advantages of the present invention as well as the structure
and operation of various embodiments of the present invention are described in detail
below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
This invention is pointed out with particularity in the appended claims. The above
and further advantages of this invention may be better understood by referring to the
following description when taken in conjunction with the accompanying drawings, in
which:
FIGS. 1A, IB, 1C, and ID show the mechanism by which one embodiment of the
present invention uses to capture, hold and release a single cell.
FIGS. 2A, 2B, and 2C show a process by which another embodiment of the
present invention uses to capture, hold and release a single cell.
FIGS. 3 A and 3B show a top-down view of the cell sorting apparatus of FIG. 2.
FIG. 4 shows an exploded view of the cell sorting apparatus of FIG 2.
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FIG. 5 shows an exploded view of yet another embodiment of the present
invention in which a cell sorting apparatus is integrated into a fluorescence-detecting
system.
FIG. 6 is the thermodynamic pressure-volume diagram for water.
FIG. 7A shows a top view of a resistor of the present invention.
FIG. 7B shows a cross-section of the resistor of FIG. 7A.
FIG. 8 shows thermal resistances as seen by a heater of the present invention.
FIGS. 9A and 9B show flow lines for flow over rectangular cavities of different
aspect ratios.
FIG. 10 shows a schematic of forces on a particle in a well.
FIG. 1 1 A shows a top view of a heater of the present invention.
FIG. 1 IB shows a cross-section of the heater of FIG. 1 1 A.
FIG. 12A shows a side view of a cell well of the present invention.
FIG. 12B shows a top-down view of the cell well of FIG. 12A.
FIGS. 13A, 13B, and 13C shows a top-down view of a silicon processing mask
set for use in the present invention.
FIG. 14 shows a top-down view of a glass processing mask.
FIG. 1 5 shows a diagram of a flow system for testing devices of the present
invention.
FIG. 16A shows a top-down view of a flow chamber of the present invention.
FIG. 16B shows a side view of the flow chamber of FIG. 16A.
FIG. 17 is a graph of pressure drop vs. flow rate for the flow chamber of FIGS.
16A and 16B.
FIG. 18A shows a top-down view of the chamber base of flow chamber of FIG.
16A and 16B.
FIG. 18B shows a side view of the chamber base of FIG. 18A.
FIG. 18C shows a top-down view of the chamber lid of flow chamber of FIG.
16A and 16B.
FIG. I8D shows a side view of the chamber lid of FIG. 18C.
FIGS. 19A-19C show a process of fabricating a glass slide of the present
invention.
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FIGS. 20A-20H show a process of fabricating a silicon wafer of the present
invention.
FIGS. 21A-21D show a process of assembling the silicon wafer of FIGS. 20 A-
20H onto the glass slide of FIGS. 19A-19C.
FIG. 22 is a graph of temperature v. resistance for platinum resistors of the present
invention.
FIG. 23 shows a configuration for a resistor testing apparatus used in the present
invention.
FIG. 24 is a graph of current v. voltage for the onset of boiling in platinum line
resistors of the present invention.
FIG. 25 is a graph of current v. temperature for the platinum line resistors of FIG.
24.
FIG. 26 is a graph of temperature v. resistance for a set of annealed platinum line
resistors of the present invention.
FIG. 27 is a graph of temperature v. resistance for a set of annealed platinum line
resistors which were heated on a hot plate.
FIG. 28 is a graph of current v. voltage for a set of annealed platinum line
resistors of the present invention.
FIG. 29 is a graph of current v. temperature for the resistors of FIG. 28.
FIG. 30 is a graph of current v. voltage for the resistors of FIG. 28 under repeated
boiling tests.
DETAILED DESCRIPTION OF THE INVENTION
FIGS. 1A-1D illustrate an exemplary system of the present invention. A cell site
10, shown in cross-section, contains a well 12 sized and shaped to hold a single cell 18.
Connected to the bottom of the well 12 is a narrow channel 14 that opens into a chamber
16 situated below the well. In this particular example, the well 12 and narrow channel 14
are etched out of a silicon wafer. The silicon wafer is attached to a glass slide on which
there is a platinum heater 20, and the alignment is such that the heater 20 is sealed inside
the chamber 16, which is filled with a fluid such as water.
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The well 12 function as a capture and hold mechanism. In operation, fluid
containing cells is flown over the top of the apparatus, and then the flow is stopped. As
shown in FIG. 1A, the cells then settle and gravitational forces will allow one cell 18 to
fall into and become trapped within the well 12. At this point the flow is started again,
5 and the cell in the well is trapped while the cells not in well are flushed away by
convection. FIG. IB shows how the well 12 is dimensioned and configured to hold only
one cell 18 within the well 12 at a time. In addition, the well 12 is configured such that
the cell 18 will not be swept out of the well due to laminar or fluid flow above.
Experiments may be performed on the trapped cells, such as by adding a rcagant.
10 When the experiments are concluded, the cells exhibiting the desired characteristics may
be selectively released from the wells. In this example, when it is desired to release cell
18 from the well 12, the operator can apply a voltage to the heating element 20 within the
chamber 16. The heating element 20 is then heated to a temperature above the superlimit
of the fluid contained within the chamber 16 to initiate vapor bubble nucleation at the
15 surface of the heating element 20, as seen in FIG. 1C. In FIG. ID, a microbubble 22 is
formed inside the chamber, creating a volume displacement. By adjusting the voltage of
the heating element 20, the operator can control the size of the microbubble 22. When
the microbubble 22 is of sufficient size, the volume expansion in the chamber will
displace a jet of fluid within the chamber 16 out of the narrow channel 14, ejecting the
20 cell 18 out of the well 12. The released cell 18 can be swept into the fluid flow outside
the well 12, to be later collected or discarded.
In another exemplary system of the present invention, the cell site 30 includes
electric field traps. Figures 2A-2C show, in cross-section, two cell sites on a substrate
such as a microfabricated chip 36. Each site includes a plurality of electrodes 32.
25 Preferably, each cell site 30 contains four electrodes, positioned in a trapezoidal
configuration, as seen in Figures 3A and 3B. The cell site 30 is configured and
positioned such that only one cell can be held within the site. The electrodes 32 create a
non-uniform electric field trap within which a single cell 34 can be held and subsequently
released. FIG. 4 illustrates how the location and polarity of the electrodes 32 can create
30 an electric field trap for capturing the cell 34.
In use, cells in fluid medium flow over the cell sites 30, as shown in FIG. 2A. By
adjusting the electric field of each electrode 32, a potential energy well can be created
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within each cell site 30. The potential energy well is of sufficient strength to capture a
single cell 34 traveling along the fluid flow and to hold the cell 34 within the center of the
trap, as seen in FIG. 2B. When the operator selects to release a cell 34, he can adjust the
electric fields of the electrodes 32 forming the trap. FIG. 2C shows how this in turn
removes the potential energy well, releasing the cell 34 back into the fluid flow. The cell
34 can then be collected or discarded.
The electrodes forming the electric field trap are preferably thin-film poles formed
of gold. This creates a three-dimensional electric field trap that is effective in holding a
cell against the laminar flow of the fluid surrounding the electrodes. Further, while only
one or two cell sites are illustrated, it is understood that the drawings are merely
exemplary of the kind of site that can be included in the cell sorting apparatus of the
present invention. The cell sorting apparatus can contain anywhere from a single cell site
to an infinite number of cell sites, for sorting mass quantities of cells. Moreover, while
the embodiments herein are described as holding cells, it is understood that what is meant
by cells includes biological cells, cellular fragments, particles, biological molecules, ions,
and other biological entities.
Because the cell sorting apparatus of the present invention allows the operator to
know the location of each cell in the array of cell sites, the operator is able to manipulate
the cells and arbitrarily sort the cells based on their characteristic under time-responsive
assays. One such method contemplates using scanning techniques to observe dynamic
responses from cells. As shown in FIG. 5, an integrated cellular analysis system 100 is
proposed in which cells are tested using light-emitting assays to determine the cell's
response to stimuli over time. The integrated system can be a microfabrication-based
dynamic array cytometer (^iDAC). The tested cells are placed on a cell array chip 110
similar to the cell sorting apparatus above, to be held in place within the plurality of cell
sites, such as those described above. Using an optical system 120 to detect fluorescence,
the response of the cells can be measured, with the intensity of the fluorescence reflecting
the intensity of the cellular response. Once the experiment is complete, the cells
exhibiting the desired response, or intensity, may be selectively released, to be collected
or later discarded. Such an integrated system would allow researchers to look at the cell's
time response.
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Any light-emitting assay in which the cell's response may vary in time is suited
for study using this proposed system. It is ideally suited for finding phenotype
inhomogeneities in a nominally homogeneous cell population. Such a system could be
used to investigate time-based cellular responses for which practical assays do not
currently exist. Instead of looking at the presence/absence or intensity of a cell's
response to stimulus, the researcher can look at its time response. Furthermore, the
researcher can gain information about a statistically significant number of cells without
the potential of masking important differences as might occur in a bulk experiment.
Specific applications may include the study of molecular interactions such as rcceptor-
ligand binding or protein-protein interactions. Signal transduction pathways, such as
those involving intracellular calcium, can also be investigated.
An advantage of the proposed integrated system is that the full time-response of
all the cells can be accumulated and then sorting can be performed. This is contrasted
with flow cytometry, where each cell is only analyzed at one time-point and sorting must
happen concurrently with acquisition. Geneticists can look at gene expression, such as
with immediate-early genes, either in response to environmental stimuli or for cell-cycle
analysis. Another large application area is drug discovery using reporter-gene based
assays. The integrated system can also be used to investigate fundamental biological
issues dealing with the kinetics of drug interactions with cells, sorting and analyzing cells
that display interesting pharmacodynamic responses. Another application is looking at
heterogeneity in gene expression to investigate stochastic processes in cell regulation.
Finally, once temporal responses to certain stimuli are determined, the integrated system
can be used in a clinical setting to diagnose disease and monitor treatment by looking for
abnormal time responses in patients' cells.
One objective of the present invention is to provide a cell analysis and sorting
apparatus which uses hydraulic forces to capture individual cells into addressable
locations, and can utilize microbubble actuation to release these individual cells from
their locations. In developing this apparatus, it was necessary to model and understand
many physical phenomena, not the least important of which includes the theory behind
bubble nucleation on micro-heaters. Further, it was necessary to design a device with the
proper dimensions so that single particles, or cells, could be held in wells against a flow.
Biological cells were not used in these experiments, as polystyrene microspheres of the
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same dimensions were thought to be more robust for testing purposes. The fabrication
process had to be designed in order to build chips with the desired attributes, and various
problems which arose needed to be resolved. Finally, it was necessary to understand the
heating of the resistors so that sufficiently high temperatures could be reached.
5 Under the theory of bubble nucleation, pool boiling takes place when a heater
surface is submerged in a pool of liquid. As the heater surface temperature increases and
exceeds the saturation temperature of the liquid by an adequate amount, vapor bubbles
nucleate on the heater. The layer of fluid directly next to the heater is superheated, and
bubbles grow rapidly in this region until they become sufficiently large and depart
10 upwards by a buoyancy force. While rising the bubbles either collapse or continue
growing depending on the temperature of the bulk fluid.
There are two modes of bubble nucleation: homogeneous and heterogeneous.
Homogeneous nucleation occurs in a pure liquid, whereas heterogeneous nucleation
occurs on a heated surface.
15 In a pure liquid containing no foreign objects, bubbles are nucleated by high-
energy molecular groups. According to kinetic theory, pure liquids have local
fluctuations in density, or vapor clusters. These are groups of highly energized molecules
which have energies significantly higher than the average energy of molecules in the
liquid. These molecules are called activated molecules and their excess energy is called
20 the energy of activation. The nucleation process occurs by a stepwise collision process
that is reversible, whereby molecules may increase or decrease their energy. When a
cluster of activated molecules reaches a critical size, then bubble nucleation can occur.
In order to determine at what temperature water will begin to boil in the
homogeneous nucleation regime, it was useful to know the thermodynamic superheat
25 limit of water. FIG. 6 is the thermodynamic pressure- volume diagram for water, which
shows a region of stable liquid to the far left, stable vapor to the far right, metastable
regions, and an unstable region in the center of the dashed curve. The dashed line is
called the spinodal, and to the left of the critical point represents the upper limit to the
existence of a superheated liquid. Along this line, Equation ( 1-1 ) holds true, and within
30 the spinodal, Equation ( 1-2 ) applies.
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^.dv) T
(1-1)
(1-2)
The van der Waals and Berthelot equations of state were used to calculate the
superheat limit of water.
[p+^y-^RT (1 . 3)
Where v is the specific volume, R is the gas constant, and a and b are constants. n=0 for
the van der Waals equation, «=1 for the Berthelot equation, and n=0.5 for the modified
Berthelot equation, a and b were computed using Equation ( 1-3 ), given the fact that at
the critical point, Equations ( 1-4 ) and ( 1-5 ) are true.
Using the above equations, the thermodynamic superheat limit of water was
computed. The results are shown below in Table I .
Equation of State
T/T cr (T cr =647°K)
Superheat Limit (°C)
Van der Waals
0.844
273
Modified Berthelot
0.893
305
Berthelot
0.919
322
Table 1. Thermodynamic superheat limit of water calculated with 3 equations of state.
These values represent the temperature above which homogeneous nucleation
must begin.
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A kinetic limit of superheat may also be computed using the kinetic theory of the
activated molecular clusters. The kinetic limit of superheat for water is about 300°C.
When liquid is heated in the presence of a solid surface, heterogeneous nucleation
usually occurs. In this regime, bubbles typically nucleate in cavities (surface defects) on
5 the heated surface. The degree of superheat necessary to nucleate a bubble in a cavity is
inversely dependent on the cavity radius, as shown in Equation ( 1-6 ).
Where T w is the surface temperature, T sat is the saturation temperature (100°C for water),
10 a is the surface tension, h, g is the latent heat of vaporization, p v is the vapor density, and
;* c is the cavity radius. For example, the surface temperature necessary to nucleate
bubbles in water with a surface that has a lum cavity radius is about 133°C. For a O.lum
cavity radius the temperature to nucleate a bubble is about 432°C, well above the highest
thermodynamic water superheat limit of 322°C.
15 Accordingly, for surfaces with cavity sizes well below lum, it is likely that
homogeneous nucleation will occur since the liquid will reach the superheat limit before a
bubble nucleates in a cavity. Micromachined surfaces tend to have very smooth surfaces.
For instance, the platinum resistors are only 3-6um wide, and 0.1 u.m thick, so it is
unlikely that cavities will exist on the surface which are large enough for heterogeneous
20 nucleation to occur. The largest likely nucleation cavity would be the thickness of the
resistor, which is 0. 1 urn, and results in a boiling temperature for heterogeneous
nucleation above the thermodynamic superheat limit as shown above. Thus, it was
assumed that homogeneous nucleation was the most likely method of bubble nucleation
to occur for the resistors of this invention.
25 However, when platinum films are annealed, thermal grooving and agglomeration
can take place at the grain boundaries. A groove will develop oh the surface of a hot
polycrystalline material where a grain boundary meets the surface. As the surface gets
hotter, the grooves deepen, initiating holes, and the platinum begins the process of balling
up in order to reduce surface area. This process is called agglomeration. The
30 agglomeration rate is insignificant at anneal temperatures below 700°C. However, for a
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600°C anneal of platinum for 1 hour, the onset of agglomeration can cause small voids in
the platinum with radii of up to about 0.5 ^m. In this case, heterogeneous nucleation
would be possible at a temperature of about 166°C.
Next, it was desirable to predict the electrical current necessary to achieve a
certain temperature of the resistor. The schematic and boundary conditions for this
resistor model are shown in FIG. 7A and 7B. For the cross-sectional slice through the
resistor (7B), the water above the heater was 450|im thick, corresponding to the height of
the silicon chamber containing the water. It was assumed that the ambient temperature
was maintained at the top of the water in the well since above this there was silicon with
water at the ambient temperature flowing over the top of it. The bottom of the glass slide
was also assumed to be at the ambient temperature since it was contacting a surface at the
ambient temperature. The resistor was about 10,000 times thinner than the glass slide and
had ohmic heating, or power generation equal to I 2 R for the entire volume of the resistor.
First, the characteristic time for the heat to conduct through the two bounding
surfaces was calculated using Equation ( 1-7 ).
T » •
a
(1-7)
Where L is the characteristic length for conduction and a is the thermal diffusivity of the
material.
Using this relation, it was found that the characteristic time for conduction
through 1mm of glass was about 2.3 seconds. Similarly, the characteristic time for
conduction through 450fim of water was 1.38 seconds. Accordingly, for this system the
time to reach steady state would be about four times greater than the highest characteristic
time, about 9 seconds. As established above, homogeneous bubble nucleation was likely
to occur, which is a molecular process and thus may be assumed to be approximately
instantaneous. The time for a bubble to nucleate was therefore far shorter than the 9
seconds necessary for the system to reach steady state, so steady state conditions are
unlikely to be achieved before the bubble nucleates.
14
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It was then necessary to determine the dominant modes of heat transfer from the
resistor to its surroundings. The purpose of this model was to predict the temperature of
the heater for a given current, before the onset of boiling. For this model, heat transfer
due to radiation was neglected.
A lumped model approach was taken for this analysis. This approximation was
checked by computing the Biot number for the resistor.
Si = — = 7jc1(T 9 «1
k Pt (1-8)
Where t is the platinum resistor thickness (0. 1 fim) and k Pt is the thermal conductivity of
platinum (7 1 .5 W/mK). It was assumed in this model a heat transfer coefficient of
h=5W/m 2 K as a high bound for natural convection. The Biot number measures the ratio
of internal conduction resistance to external convection resistance. Since the Biot
number was much less than unity, the lumped body approximation was used and an
assumption was made that the entire resistor was at a uniform temperature.
FIG. 8 shows the thermal resistances between the resistor and the ambient
temperature. For the purpose of this order of magnitude estimate of the heat transfer
mechanisms, steady state conditions were used in determining thermal resistances. First,
the thermal resistance due to convection through the water was computed. For this case it
was assumed there was natural convection since the water above the heater was stagnant,
and boiling was not occurring. The thermal resistance due to convection was calculated
below.
= 7TT = ttt = 667x107 £
hA hwL W ( 1-9 )
Where w is the resistor width (3|im) and L is the resistor length (lOOOjam).
Next the thermal resistance due to conduction through the platinum resistor, glass
slide, and water were computed. The resistance due to conduction was given by:
15
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D
conduction
L_
kA (1-10)
Where L is the length through which heat conducts, and A is the cross-sectional area. For
the platinum, the length through which heat conducts was very long (12mm) and the
cross-sectional area was very small, resulting in a high thermal resistance:
5
RvtoUnun, = = 5.4*1 0* —
plenum k ^ (w w (| .n,
Where / is the platinum film thickness (0.1 fim), L P( is the length through which heat
conducts (12mm), w is the width of the resistor (3[im), and k Pt is the conductivity of
platinum (71 .5W/mK). Similarly, the thermal resistances of the glass and water were
10 computed.
glass
water
k g Lw
= 4.LdO
W
— — = 2.2jc10 5 —
kLw W
( 1-12 )
(1-13)
Where L g is the length of glass through which heat conducts (1mm), k g is the conductivity
of glass (0.81 W/mK), L is the length of the resistor (lOOO^rn), w is the width of the
15 resistor (3jim), L w is the length of water through which heat conducts (450|am), and k w is
the conductivity of water (0.67W/mK).
From this it was shown that Rgi aS s and R W ater were the dominant thermal resistances
for the system. Thus, heat transfer due to convection in the water and conduction through
the platinum were negligible.
20 An estimate the temperature of the resistor as a function of time for a given
current using semi-infinite body theory was then made. For small times (t<lms) it was
assumed that both the water and glass are semi-infinite bodies with initial temperature T a .
At t=0, a constant heat flux (due to the resistor) is applied at the water-glass interface
(x=0). The one-dimensional temperature profile was computed using the infinite
16
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composite solid solution. The region x>0 is water, x=0 is the resistor, and x<0 is the
glass, A one-dimensional model was used for short times since the length of the resistor
(L=1000u.m) was much less than the width of the resistor (L=6|im). The temperature was
assumed to be constant along the resistor, and lateral conduction was neglected for small
5 times. This model will break down when the lateral conduction becomes significant, and
when the assumption of semi-infinite bodies becomes invalid. The boundary conditions
for this problem are given below.
7; = T 2 ,x - 0,/ > 0
K x K 2
</, +</: = H
,.y = 0,/ >0
d-14)
(1-15)
(1-16)
10 Where K is the thermal conductivity (0.61 W/mK for water and 0.88 W/mK for glass), q
is the heat flux, and the subscript 4 1 * denotes water, and K T denotes glass.
The solution for the temperature profiles in water and air for a constant heat flux q
(W/m 2 ) applied at x=0 is given by Equations ( 1-17 ) and ( 1-18 ).
~ rp 2qyja,a 2 t x
Ti ~ T <> = ^ I— v i — ier ^ c VT = d-17)
15
2q^a l a 2
K ] ^ja 2 ~ + K 2 Ja~ { lyfcrj
Where a is the thermal diffusivity (1.47x1 0" 7 m/s 2 for water and 4.4x1 0~ 7 m/s 2 for glass)
and T 0 is the initial temperature of the body.
The solution was also used to check the semi-infinite body assumption. For times
equal to or less than 1ms, and a reasonable heat flux such as 2.5x1 0 7 W/m 2 , the heat
20 penetration depths into the glass and water were less than lOOfirn. The total thickness of
the water was 450^m and of the glass was 1mm, so the semi-infinite body assumption
17
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held true. The one-dimensional model was sufficient for determining the temperature of
the resistor at small times.
Using the theory described above, it was possible to predict the power necessary to
form a bubble. Since homogeneous bubble nucleation was assumed, the bubbles would
5 form at approximately the superheat limit of water. The value of 305°C given by the
modified Berthelot equation (Table 1) was used. Next, the infinite composite solid
solution was used to calculate the temperature of the heater for a given time, say 1ms.
Rearranging equation ( 1-17 ) to solve for the heat flux, or power per unit area at position
x=0, it was derived:
10
P (K x fe + K 2 fc\T-T 0 )
Lw 2^7t < , - 19 >
For an initial temperature of 20°C, and the other properties given above, the heat
flux necessary to heat the resistor to 305°C in 1ms was computed from ( 1-19 ) to be
1.32xl0 7 W/m 2 . For typical resistor dimensions of w=6jam and L=1500|am, the
15 necessary power was about 120mW.
The micromachined wells must be of the proper dimensions to ensure that
particles which settle into them remain held in the wells once a flow above them is
initiated. The theory of slow viscous flow over cavities has been well characterized and
the streamlines for various geometries have been calculated and experimentally verified.
20 FIG. 9 shows the flow pattern for laminar flow over a rectangular cavity for two
different width to height aspect ratios. From these flow patterns it was seen that there
was a separating flow line which penetrates slightly into the cavity. Below this line there
were one or two vortices, depending on the aspect ratio of the cavities. A particle below
the separating flow line would not be swept out of the cavity by a slow flow in the
25 laminar range, though the vortex may agitate the particle.
An order of magnitude calculation was performed in order to compare the relative
sizes of the gravity force pulling a particle down, compared to the viscous shear force
pulling a particle out of the well. A diagram of a particle in a well with flow over the top
is shown in FIG. 10.
18
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The force of gravity acting on the particle was dependent on the difference in
density between the particle and the water, Ap. The density of water is approximately
1000kg/m 3 , and the density of the polystyrene beads used in the experiments was given
by the manufacturer as 1060kg/m 3 . The density of cells ranges from 1050-1 100kg/m 3 .
5 Accordingly, the force of gravity, F g was computed as shown:
Where a is the particle radius (5xl0~ 6 m), and g is the gravitational constant.
The viscous shear force acting on the particle was computed by assuming the top
10 of the particle was at the top of the well, and that the flow profile was parabolic. The
shear stress at the wall was:
du
Where p is the viscosity of water (lxl0" 3 kg/ms) and u(y) is the velocity profile as a
15 function of.y, the distance from the wall.
Assuming a parabolic velocity profile in the flow chamber, the flow profile was
calculated for a known chamber height and volume flow rate.
u(y) = ^-y(h-y) ( ,. 22)
wh
U M = ^ y{h - y) d-24)
du 6Q
dy^ ^ d-25)
20 Where V is the average flow velocity, w is the chamber width, and h is the chamber
height.
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The viscous shear force on the cell was estimated as the wall shear stress
multiplied by the area being effected, approximately na 2 .
F — r na 1 — u. ^ na 2
Where a is the cell radius. Finally the ratio of gravity to viscous force was computed.
4
F* A^3«]g lApa&vh 2 (1-27)
F t 6Q 2 9mQ
wh
Using the flow chamber dimensions in FIG. 1 6, and a range of reasonable flow
rates, this ratio was computed.
e = 1 JfLfK=2.1^U^ = 292
minv ^ / F ¥
e = 10 ifLfF = 21^U^ = 29
minv s ) F v
e = ioo^fF = 2io^U^ = 3
mini, s J F v
It was necessary that the ratio of forces be greater than one so that the gravity
force was stronger than the viscous force. These numbers were used to aid in
determining a range of acceptable operating flow rates.
Another relevant piece of information was the time it took for the particles to
settle. At low Reynolds number, an isolated rigid spherical particle will settle with its
Stokes velocity.
u<} = 2a 2 (p s -p)g
9/J d-31)
20
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Where a is the sphere radius (5|im for a polystyrene bead), p s is the density of the bead
(about 1060kg/m 3 ), p is the density of water (1000kg/m 3 ), and ju is the viscosity of water.
Using these values a Stokes velocity was calculated as:
V =5x\Q- f '^- = 5^-
S S ( 1-32 )
Using this velocity to check the associated Reynolds number it was found that
Thus, the assumption of low Reynolds number was valid. The Reynolds number
is the ratio of inertial effects to viscous forces. For this case, only the highly viscous
regime applied and inertial effects were negligible.
Another value which was checked was the Peclet number. This is the ratio of
sedimentation to diffusion. For the particles to settle, the Peclet number must be
sufficiently high, otherwise the particles will diffuse throughout the liquid.
aU°
Pe =
O 0 < 1-34 )
D° = kT =4*1(T' 4 —
67T/M2 s * 1-35 )
^ = 6.rl0 : »1 ( 1-36)
Where D° is the Brownian diffusivity, and k is the Boltzmann's constant
(1.381xl0 16 erg/cm). Thus the Peclet number was sufficiently high for settling to
dominate over diffusion.
The value calculated above for the Stokes velocity is that for an isolated particle;
however, in the case at hand there were many beads settling at once. This was taken into
account in the calculation of the hindered velocity. A function of the particle volume
fraction is multiplied by the Stokes velocity to result in the hindered velocity of particles
in the suspension.
21
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f(<fi) = (\-(f>) 5] =0.95 ^- 38 >
*m urn ( !' 39 )
= 4.75*1 0" 6 — = 4.75^-
5 ^
Where <p is the particle volume fraction (about 0.01 for this case). Accordingly, the time
necessary for all the particles to settle to the bottom of the flow chamber was calculated
using the hindered velocity and the chamber height, the maximum distance to be traveled.
5
t = — = 1665 = 2.76min „ aA v
5 U ( 1-40 )
Where h is the chamber height (790^m). This settling time was used as a guideline in
experiments.
A more reasonable assumption for calculating the settling time was that the
10 distance the particles fell is an average of half the chamber height. For this case a settling
time of about 83 seconds was obtained.
For the given pressure increase associated with the bubble formation in the large
sealed well, the flow rate out of the channel in the top of the well was calculated. Since
the Reynolds number was in the creeping flow regime (Re<l), inertial effects neglected,
15 and the initial, instantaneous flow out of the channel was computed using the steady state
equation for flow through a circular aperture at low Reynolds number.
(1-41)
Where Q is the volume flow rate, AP is the pressure drop, c is the aperture radius (-2.5 or
20 4fim), and fi is the water viscosity.
Since the pressure change due to the bubble formation was not easily calculable,
the volume flow rate out of the chamber was estimated in a different way. Because water
is incompressible, it was assumed in the model that the bubble formation as a volume
injection into the chamber resulted in the same volume being ejected from the chamber
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over the characteristic bubble formation time. For instance, if it took 1ms to form a
lOjim diameter bubble, then the resulting volume flow rate out of the chamber was
calculated as follows.
K = |^=5.24.vlO-'W (M2)
2 = 7 = 5.24*10-'^ (MJ>
5
Using the volume flow rate the average velocity of fluid out of the channel was
calculated, and it is seen that the Reynolds number of the flow was indeed low.
rr Q nim
V - = 27
nc 2 s
Re = ^ = 0.067<l ( ^ 5)
10 Where c is the channel radius (2.5um). The force of the fluid jet on the particle was
calculated using the Stokes drag force:
F D =b7TfjaV = 2.5x1 0" lJ /V
( 1-46 )
Where a is the radius of the spherical particle (5|am for polystyrene beads). Comparing
15 this to the gravitational force ( 1-20 ) pulling the particle down, it was found that the force
of the jet on the particle was much greater than the force of gravity.
F t = Ap^a'g = 3.U10-" N « F D ( ^ }
F g a 2 (»-")
23
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Where Ap is the difference in densities between the water and the polystyrene beads (60
kg/m 3 ). It was seen that as the particle radius increased, the effect of gravity increased.
For typical cells, the radius ranges from 5u.m (red blood cells) to 20um (most other cells)
to lOOj-im (embryos and eggs). This device will most likely be used for cells on the order
5 of 5-10um in radius so the above calculation was representative of the expected
applications.
DESIGN OF THE COMPONENTS
10 A. RESISTIVE HEATERS
In order to heat the water to a sufficiently high temperature for microbubble
formation, resistive heaters were used. The heaters were made of thin-film platinum on
standard glass slides, in designing the heaters it was necessary first to determine a range
of resistances and currents to attain the desired power output. The design constraint for
15 this step was the need to keep the current density below the electromigration limit of
platinum, while retaining an adequate degree of ohmic heating. The electromigration
limit is the maximum current density which platinum can endure before the atoms begin
to migrate leaving the resistor inoperable.
20 necessary to design the resistors to operate at a current density below this limit.
Where R is the resistance (Q), L is the length of the resistor (m), t is the film thickness
25 (m), w is the width of the resistor (m), and p is resistivity of platinum (Qm).
The power output of a resistor is a function of the current and resistance, as shown
below.
The electromigration limit of platinum was reported to be J=9xl0 6 A/cm 2 . It was
The resistance of a line heater is calculated as follows.
(1^9)
P = I 2 R
(1-50)
24
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y = _L <9jc io 6 -A
wt
cm
( 1-51 )
Where / is the current (A) and J is the current density.
Accordingly, as the currents were limited by the electromigration limit, the
resistances needed to be sufficiently high to achieve the desired power output. The power
5 output necessary to form a bubble was estimated by using the numbers from Lin et al.'s
paper, * Micro bubble Powered Actuator', herein incorporated by reference, where
microbubblcs were formed on a polysilicon line heater. Their resistor was on top of a
thin dielectric layer, which was on a silicon wafer. It was reasonable to assume that the
heat dissipation of this configuration might well be greater than the heat dissipation of the
10 platinum line resistor fabricated on a glass slide. Also, a liquid with a higher boiling
necessary to nucleate bubbles under these conditions was approximately 65mW.
Slide Name
Resistor
Length (urn)
Width (um)
Resistance (Ohms)
Electromigration Limit (mA)
Max Power (mW)
Slidel
1
3000
3
1000
22
467
2
2500
3
833
22
389
3
500
3
167
22
78
4
1000
3
333
22
156 !
5
1000
4
250
29
207
6
2000
3
667
22
311
7
1500
3
500
22
233
8
1000
5
200
36
259
Slide2
1
300D
3.6
483
22
226
2
2500
3.6
400
22
187
3
500
3
167
22
78
4
1000
3,6
150
22
70 i
5
1000
6
167
43
311
6
2000
3.6
317
22
146
7
1500
3.6
233
22
109
8
1000
3.5
180
22
84
SHde3
1
3000
6
625
43
1166
2
2500
6
521
43
972
3
500
3
208
22
97
4
1000
3
417
22
194
5
1000
6
208
43
389
6
2000
6
417
43
778
7
1500
6
313
43
583
8
1000
6
208
43
389
Table 2. Resistor dimensions, resistances, and electromigration limits.
15 Using this as a guideline, the resistances were chosen to range from 167Q-1000Q,
yielding maximum powers before electromigration of 70-1 166m W. These powers were
chosen to be up to an order of magnitude greater than necessary to avoid reaching the
electromigration limit in the operation of the resistors.
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The resistivity of platinum actually varies with temperature and film deposition
conditions, but for these calculations it was taken to be lxlO" 7 Qm. This is the value for
bulk platinum, however the resistivity of thin film platinum can vary widely. Heater
widths range from 3 -6jam and lengths range from 500 -3000jim. Some heaters were
designed to have a narrow region, 100|am long in the center, which would be hotter than
the rest of the resistor. FIG. 1 1A is a top view of a heater configuration, while FIG. 1 IB
shows a cross-sectional view of the heater and its dimensions. A table of resistor
dimensions, maximum currents, and maximum power outputs is also shown in Table 2.
The lines connecting the contact pads to the heaters were designed to have a far
lower resistance than the heaters. This was done to ensure that the lines did not heat up,
and that they remained approximately at the ambient temperature. The connector line
widths were chosen to be 1500|am with lengths of 12mm. The total resistance of each
line was about 7.7Q.
B. WELLS
Square wells were micromachined into silicon in order to hold cells. It was
necessary to choose a range of dimensions for these wells to allow for tests with different
particle sizes and flow rates. The final goal was to have the ability to trap one particle in
each of an array of wells.
Chip Number
Well Dimension (urn)
Hole Dimension (um)
1
16
5
b
16
8
2
10
5
2b
10
8
I 3
20
5
3b
20
8
4
30
5
4b
30
8
5
40
5
5b
40
8
6
50
5
6b
50
8
Table 3. Well Dimensions
Side lengths of the wells were chosen to range from lO^im, corresponding to the
smallest test bead size, up to 50u.m. Well sizes ranging from 10-50fxm were chosen.
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Narrow channel widths of 5jim and 8jim were chosen since both these sizes are smaller
than the minimum test particle size of lO^im and it is necessary that particles not be able
to settle down into the narrow channel. The table of well dimensions is shown in Table 3.
A diagram of the well geometry is shown in FIG. 1 2 A, which shows a side view, while
5 FIG. 12B shows a top-down view.
Photomasks for use in the device fabrication were created using standard mask
layout software. The mask set for the silicon processing are shown in FIGS. 13A-13C
and the glass mask set is shown in FIG. 14A and 14B.
Three masks were designed for the silicon portion of the device processing. One
10 mask was created for the cell wells (FIG. 13 A), one for the narrow channels within the
wells (FIG. 13B), and one for the large wells (FIG. 13C) etched from the backside of the
wafer to enclose the heaters. Two masks were made for the fabrication of the platinum
heaters on the glass slides. One mask (FIG. 14) was designed to pattern the metal.
In order to test the finished devices, a fluidic system as illustrated in FIG. 15 was
15 designed and assembled. A syringe pump 1 50 was used as the flow source for the bulk
fluid, and flow rates ranging from 1 to 100 ^L/min were specified. Beads, cells, or cell
stimuli were injected through the sample injection valve 152. A pressure sensor 1 54 was
located before the flow chamber 156 so that the pressure drop across the chamber could
be monitored. All fluid was outlet into a waste beaker 158 which could be reused if
20 desired.
A schematic of the flow chamber 156 is shown in FIGS. 16A and 16B. The flow
chamber was machined from plexiglass so that it was clear and a microscope was used to
observe cell behavior from above the chamber. HPLC (high-performance liquid
chromatography) fittings were used with tube dimensions of 1/16 inch outer diameter and
25 0.020 inch inner diameter. The gasket between the slide and the top cover were made
from PDMS (poly dimethyl siloxane), a flexible polymer. A seal was formed by
screwing the top plate down onto the bottom plate. Aluminum molds were machined in
order to create PDMS gaskets of the proper dimensions. Gaskets were compressed until a
hard stop was reached. The stop was provided by the spacers, made of metal shim stock,
30 in order to accurately specify the channel height. The aspect ratio of the channel's width
to height was greater than 10, allowing the assumption of a parabolic velocity profile-
plane Poiseuille flow.
27
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5
The height of the flow chamber was 790|im (determined by thickness of metal
spacer). Flow rates ranged from 1 to 100 jaL/min and corresponded to Reynolds numbers
of 0.00 1-0. i. In this creeping flow regime, the entrance length for fully developed flow
was calculated to be negligible. These calculations are shown below.
min A c s (1-52)
™ /* S d-53)
hV .
Re mm =—=. = 0.0011
hV
RC_ =— =5- = 0.11
(1-54)
(1-55)
AT * hRe ™* = 2.6pm
30 M (1-56)
Where K mjn is the minimum average velocity, Q min is the minimum volume flow rate
(l^iL/min), A c is the cross-sectional area of the channel (h=790jim, w=12mm), V min is the
maximum average velocity, (2max is the maximum volume flow rate (lOOjaL/min), Re is
10 the Reynolds number, v is the kinematic viscosity of water ( 1 x 1 0" 6 m 2 /s), andX e is the
entrance length for fully-developed flow.
Electrical connections to the contact pads were made using a probe station.
Contact pads were positioned outside of the PDMS gasket and were thus kept outside of
the fluid flow.
15 In order to ensure the proper flow characteristics of the flow chamber, dye was
injected into the flow and the resulting profile was observed. The results were used to
discover problems such as blockages in the flow chamber and correct them. When a
uniform flow was established, 10|im diameter beads were injected into the flow and
observed under a microscope.
20 The pressure drop across the flow chamber was monitored using a pressure
transducer. The majority of the pressure drop was caused by the connector tubing, but by
comparing the pressure reading to the theoretical value, the presence of bubbles and other
blockages to the flow may be detected.
28
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5
The pressure versus flow rate plot for the flow chamber is shown in FIG. 17. The
theoretical value is plotted with the experimental measurements. When these two values
do not match, a blockage in the chamber or tubing is probable.
The pressure drop through the tubing was calculated using the following equation.
A/> = ^A*
Where /a is the viscosity of water (lxl0~ 3 kg/ms), r is the tube radius (0.254mm), and Ax is
the tube length (m). The pressure drop through the chamber was calculated to be
negligible in comparison. The flow chamber schematic with dimensions is shown in
10 FIGS. 18A-18D.
FABRICATION OF THE COMPONENTS
The platinum heaters were fabricated on standard lx3in glass slides using a lift-
15 off process. The process flow is shown in FIGS. 19A-19C. In the first step illustrated as
FIG. 19 A, photoresist was spun onto the glass slide, exposed using mask 4, and
developed. Next. 100A of titanium and 1000A of platinum were evaporated onto the
slide, as seen in FIG. 19B. The titanium served as an adhesion layer between the glass
and the platinum. In the following step, the slide was submerged in acetone to dissolve
20 the photoresist and lift away the metal which was deposited on top of the photoresist, as
depicted in FIG. 19C. Only the platinum resistors were left on the glass slide. Some
slides were then annealed in a tube furnace at 600°C for 1 hour. While not used in this
example, it is contemplated that photoresist may be applied manually to the slide to attach
the silicon chip to the slide.
25 The silicon chip process flow is shown in FIGS. 20A-20H. Double Side Polished
(DSP) four inch diameter silicon wafers were used. In the first step shown as FIG. 20A,
1 urn of thermal oxide was grown on the wafer. Next the oxide was patterned using mask
1, FIG. 20B. Resist was spun on top of the oxide and patterned using mask 2. The
resulting configuration was called a nested mask, shown as FIG. 20C.
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First the photoresist mask was used to etch the narrow 5 urn trenches, then the
oxide mask was used to etch the cell wells, as shown in FIGS. 20D and 20E. Next the
wafer was turned over and photoresist was deposited and patterned on the back side using
mask 3 (FIG. 20F). A deep silicon etch was then performed to etch through the wafer
5 and intersect the narrow trenches etched previously (FIG. 20G) to obtain a finished wafer
(FIG. 20H).
A complete device consisted of a silicon chip attached to a glass slide by
photoresist, as shown in FIGS. 21C and D. The resist provided a water-tight seal so that
volume expansion in the bubble wells resulted in a burst of fluid being pushed through
10 the narrow channel and ejecting a cell.
To facilitate the assembly process, alignment marks were fabricated on the glass
slide and matching holes were etched in the silicon chip. The alignment tolerances were
sufficiently large (about 2mm) that the chip could be aligned to the slide by hand using
just the naked eye, while still positioning the bubble wells over the platinum heaters.
15 Photoresist was painted onto the silicon chip around the bubble wells using a
toothpick. Drops of water were deposited into each well using a pipette, then the glass
slide was visually aligned from above and stuck down onto the chip. The drops of water
served to fill the bubble wells and get pushed through the narrow channel to fill it with
water. The device was now ready to be tested in the flow chamber.
20 Next, the resistance of the platinum resistors were studied. The film thickness was
first measured using a profilometer. The platinum thickness measurements ranged from
about 800-900A, so the average value of 850A was used in the subsequent calculations.
The resistance along metal lines wide enough not to be strongly affected by variation of a
few microns was measured using a multimeter. The lines used for this measurement were
25 measured in an optical microscope to be about 1510u.m wide. The length of the lines was
about 8mm. Knowing the width, thickness, and length of these lines, as well as the
measured resistance, the resistivity of the thin film platinum at room temperature was
determined. The measured resistance was 1 5Q, and the computed resistivity was
calculated below.
30
p = — = 2A\x\0- 1 Cbn
L (1-58)
30
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Where t is the film thickness (850A), w is the line width (15 13^xm), R is the measured
resistance (15Q), and L is the length of the line (8mm). This resistivity was more than
twice the value for bulk platinum (lxlO" 7 Qm), but was a reasonable value for thin film
5 platinum. This is because bulk platinum is a crystalline material, whereas thin film .
platinum is polycrystalline and the grain boundaries significantly increase resistance.
Next, the resistance of the resistors was measured with a multimeter. Using the value
of resistivity from above, the line width of each resistor was determined. The line widths
were also measured using an optical microscope to an accuracy of about ±1 jim. The
10 results of this measurement for two different resistor slides are shown in Table 4.
Resistor #
L (urn)
R (Ohms)
Computed Line Width (urn)
Measured (urn)
Design (um)
Slide 1
1
3000
1020
8.34
8
3
2
2500
845
8.39
8
3
3
500
185
7.66
8
3
4
1000
347
8.17
8
3
5
1000
272
10.42
10
4
6
2000
672
8.44
9
3
7
1500
504
8.44
8
3
8
1000
260
10.90
10
5
Slide 3
I 1
3000
850
10.01
10
6
2
2500
728
9.74
10
6
3
500
247
5.74
6
3
4
1000
479
5.92
6
3
5
1000
316
8.97
9
6
6
2000
620
9.15
9
6
7
1500
450
9.45
10
6
8
1000
270
10.50
10
6 !
Table 4. Resistance measurements and calculated, measured, and designed line widths.
15 From this it was determined that the measured and calculated line widths were within
the range of error for the measurements, confirming the resistivity calculation. The
resulting plot of normalized resistance versus temperature is shown in FIG. 22. The
resistance was normalized using the resistance at room temperature. This curve was used
later to predict the temperature of a resistor, knowing the resistance at room temperature
20 and measuring the resistance during operation.
Using the cross-sectional area of the resistors, the maximum current before
electromigration was calculated. It was known that maximum current density before
electromigration is 9xl0 6 A/cm 2 . Using this the maximum current for each resistor was
calculated. The results of this are shown in Table 5.
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Resistor &
IVICIAIIIIUIIl villi vlll ^HIA%J
Slide 1
o.o
71 1
r 1 .O
2
ft &
71 7
O
7 7
cc c
Oj.j
4
8.2
69.9
5
10.4
89.1
6
8.4
72.1
7
8.4
72.1
8
10.9
93.2
Slide 3
1
10.0
85.6
2
9.7
83.2
3
5.7
49.1
4
5.9
50.6
5
9.0
76.7
6
9.1
78.2
7
9.5
80.8
8
10.5
89.8
Table 5. Computed electromigration limits for resistors.
5 These results were used as guidelines during testing of microbubble devices to
avoid burning out the resistors.
The main objective for the resistors was that they be able to reach high enough
temperatures to boil water. The resistors were tested on a probe station using an
HP4145b to vary the voltage and measure the resulting current through the resistor. A
10 PDMS gasket was placed on top of the slide and filled with water. The gasket contained
the water and kept it from touching the electrical contacts and probes. FIG. 23 is a
schematic of this configuration.
Upon ramping the voltage across resistors from zero to about 20-30 V, there was
violent bubbling originating not from the hot part of the resistor, but from the edges of the
15 wide connector lines. It was evident that the bubbles were gas bubbles and not water
vapor bubbles because the bubbles did not condense when the heater was turned off.
Further experimentation revealed that electrolysis of the water was occurring and the
water was being broken down into hydrogen and oxygen. After flushing the slides,
gaskets, and glassware for several minutes with deionized water, and testing again, the
20 problem of electrolysis was eliminated.
When the problem of electrolysis was eliminated, the resistors were once again
tested in water. When the resistor reached a sufficient temperature, boiling occurred
along the length of the heater. After the power was turned off, small air bubbles
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remained on the resistor due to the dissolved gas coming out of solution, as described
previously. In subsequent tests, the air bubbles served as nucleation sites for boiling, the
inception of boiling occurred at a much lower temperature. When boiling begins and
bubbles form on the resistor, the heat dissipation into the water increases drastically. This
is a favorable phenomenon for the operation of the device because the onset of boiling is
represented as a sharp increase in current on the I-V curve. This is because when the heat
dissipation increases, the temperature decreases, resulting in a lower resistance and thus a
higher current through the resistor. An I-V curve for the onset of boiling on a line resistor
is shown in FIG. 24.
In this I-V curve it is shown that for the first run when no bubbles were present on
the line, there is a sharp jump in current at the onset of boiling. For the second run,
residual bubbles were left on the heater and served as nucleation sites for boiling resulting
in a smooth I-V curve with boiling beginning at a lower temperature. The two curves are
very close after the boiling begins for run 1.
In later tests, when no dissolved gas came out of solution, the jump in the I-V curve
occurred during each heating cycle for the resistors, since there were no residual air
bubbles left when the power was turned off.
Using the calibration given in FIG. 22 for the temperature-resistance relationship of
the resistor, the temperature of the resistor for each current was plotted to find the boiling
temperature. The current vs. temperature plot corresponding to the I-V curve shown
above is in FIG. 25. On this plot water is shown to boil at approximately 308°C, at which
point the temperature drops rapidly due to the increased convective heat transfer
associated with boiling.
The boiling points for the 5 resistors tested ranged from 250°C to 308°C. The
lowest calculated value for the superheat limit of water was found to be 273°C, so these
measured boiling points suggest that the bubble nucleation occurs either in the
homogeneous regime, or by a weak heterogeneous mechanism.
After a considerable amount of testing of the resistors characterized above, a drift in
the boiling temperature became apparent. In order to determine the reason for this, the
resistors were recalibrated as described in the previous section. The temperature versus
normalized resistance curve is shown in FIG. 26. The dramatic change in temperature-
resistance characterization led to the testing of a second generation of resistors. It is
33
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thought that these changed characteristics are caused over time by the heating of the
resistors. The operation of the resistors effectively caused them to anneal themselves.
Annealing changed the geography of the platinum grain boundaries and thus changed the
resistivity of the resistors.
5 In order to avoid this effect in future testing, new resistor slides were annealed at
600°C for 1 hour as the last step in their process. This temperature is higher than
operating temperatures are likely to reach, but not so high that major agglomeration will
result. Once the anneal was complete, the new resistors were characterized as described
above for the first generation resistors.
10 First, the resistivity of the platinum at room temperature was found to be
2.056x 10" 7 Qm, less than the unannealed resistors that were 2.41xlO" 7 Qm. Next the
resistances were measured using a multimeter, and the line widths were computed as
before, as shown in Table 6.
15
Resistor #
t(urn)
R (Ohms)
Computed Line Width (um)
Design (um)
Slide 3
1
3000
553
13.12
6
2
2500
481
12.57
6
3
500
146
8.28
3
4
1000
281
8.61
3
5
1000
205
11.80
6
6
2000
409
11.83
6
7
1500
310
11.70
6
8
1000
186
13.00
6
Tabic 6. Measured resistances and computed line widths of second generation resistors.
The temperature-resistance characteristic or the resistors was then measured on a
hotplate as described above, and is shown in FIG. 27.
20 At this point, the bubble formation characteristics of the resistors were tested as
described previously with boiled, deionized water. Voltages were ramped up by 0.5V
steps with delay times of 1ms using the HP4145b, as before. None of these tests resulted
in residual gas bubbles since the delay time was short, and the maximum voltage used
was just above the bubble nucleation voltage, determined by testing. All resulting vapor
25 bubbles condensed back into the liquid phase within one minute of stop of current flow.
A resulting I-V curve is shown in Figure 28, and the corresponding temperature
curve is shown in Figure 29. From the curve we can see that the onset of boiling
occurred at about 200°C, a much lower temperature than for the first generation resistors,
34
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and well below the superheat limit of water. For the 8 second generation resistors tested,
boiling points ranged from 128°C-200°C, with the majority of the temperatures above
180°C. This suggests that the boiling is in the heterogeneous nucleation regime as
discussed earlier. The cavity radii corresponding to these boiling inception temperatures
5 are calculated from Equation ( 1 -59 ).
2oT
p _ SOI
C h fgP XT w 'T sat ) (1-59)
The results of this calculation are shown in Table7.
10
Resistor #
Boiling Temperature (C)
Cavity Radius (um)
1
200.7
0.33
2
198.3
0.34
3
170.4
0.47
4
183.2
0.40
5
128
1.19
6
188
0.38
7
189
0.37
8
169
0.48
Table 7. Bubble nucleation cavity radii corresponding to measured boiling temperatures.
From this we can see that bubbles were nucleated in radii ranging from 0.3-
1.2jim. As discussed previously, these cavities were most likely formed during the
15 600°C anneal, during which the grooves at the grain boundaries widened creating
cavities.
The second generation resistors were also tested for the repeatability of their boiling
temperatures. I-V curves were measured as in the previous section, and then remeasured
for the same conditions several times. Between measurements, time was given for the
20 vapor bubbles to dissipate so that the characteristic jump in the I-V curve at boiling could
be observed with each measurement. The boiling point was found to be very repeatable,
and an example of the results is shown in Figure 30. This result demonstrated the
potential of a control system based on a jump in the I-V curve at the onset of boiling,
since the boiling point remained fixed.
25 Another interesting result from this testing is that for a particular resistor, the
bubbles tended to nucleate in the same locations on the resistor each time. This
35
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strengthens the hypothesis that the bubbles are nucleating in the heterogeneous regime, in
cavities created by thermal grooving caused by the annealing.
RESULTS
The cell chip was attached to the glass resistor slide as described earlier, and then
tested in two ways. First tests were done with stagnant fluid on the device. Then the
device was put into the flow chamber for testing. The results of these tests are described
below.
For these tests, several drops of bulk solution were placed on top of the cell chip,
and contained by the PDMS gasket. A drop of the polystyrene bead solution was then
added to the bulk fluid and allowed to settle. The bulk solution was a 0.05% solution of
Triton x-100 surfactant in deionized water. The bead solution was about 1% beads
diluted in the same bulk solution. Some of the beads settled into wells, as shown in
Figure 42. When voltage across the resistor was ramped up by the HP4145b, an I-V
curve with a jump similar to that in FIG. 24 was produced, demonstrating that boiling had
occurred. Consequently, the bubble formation under the well caused a volume expansion
which rapidly ejected the beads from the well. First the beads are in the well, and then
they are rapidly expelled. This sequence was also captured on videotape, and the process
was repeated multiple times with the same success.
Preliminary dynamic testing was performed in the flow chamber. Beads were
ejected in a similar way to the static test, and carried away in the flow. The preliminary
tests suggested that the beads are held in the wells against a reasonable flow rate, and are
ejected into the flow when a microbubble forms.
While the invention has been particularly shown and described above with
reference to several preferred embodiments and variations thereon, it is to be understood
that additional variations could be made in the invention by those skilled in the art while
still remaining within the spirit and scope of the invention, and that the invention is
intended to include any such variations, being limited only by the scope of the appended
claims.
36
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What is claimed is:
1 . A cell sorting apparatus for manipulation of cells comprising:
an array of geometric sites arranged across a substrate in a defined pattern,
5 each site being dimensioned and configured to hold a single cell, wherein each
site includes a capture mechanism that is capable of selectively capturing the
single cell, and further wherein each site includes a release mechanism to
selectively release the single cell from the site.
2. The apparatus of claim 1, wherein each site has a unique address and is
10 independently controllable with respect to another site.
3. The apparatus of claim I, wherein each capture mechanism comprises a well.
4. The apparatus of claim 3, wherein each well is sized and shaped to hold only the
single cell.
5. The apparatus of claim 4, wherein the single cell is held inside the well by gravity.
15 6. The apparatus of claim 4, wherein the well has an inner diameter ranging from
about 10 to 50 microns.
7. The apparatus of claim 3, wherein each well is connected by a narrow channel to a
chamber located below the well.
8. The apparatus of claim 7, wherein the narrow channel has a width of about 5 to 8
20 microns.
9. The apparatus of claim 7, wherein the release mechanism comprises an actuator
disposed within the chamber.
10. The apparatus of claim 8, wherein the actuator comprises a heating element.
11. The apparatus of claim 10, wherein activation of the heating element induces
25 bubble nucleation, creating volume expansion within the chamber to eject the cell
out of the well.
12. The apparatus of claim 1 1, wherein the heating element comprises two wide low-
resistance lines connected by a high-impedance line resistor.
13. The apparatus of claim 1 1, wherein the wide low-resistance lines are about 12 mm
30 long and about 1 .5 mm wide.
14. The apparatus of claim 13, wherein the total resistance of each line is about 7.7
Ohms.
37
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15. The apparatus of claim 14, wherein a surface temperature of the heating element
is above a superheat limit of a liquid inside the chamber to induce bubble
nucleation.
16. The apparatus of claim 12, wherein the resistor is formed from platinum.
5 1 7. The apparatus of claim 16, wherein the resistor is about 3-6 microns wide, and
about 500-3000 microns long.
18. The apparatus of claim 17, wherein the resistor has a roughened surface to induce
bubble nucleation.
19. The apparatus of claim 18, wherein a surface temperature of the resistor is
10 sufficient to induce bubble nucleation.
20. The apparatus of claim 19, wherein the surface temperature of the resistor is about
100°C to about 280°C
21 . The apparatus of claim 16, wherein the resistor contains at least one hole for
inducing bubble nucleation therein.
15 22. The apparatus of claim 1 1, wherein the bubble is about 200 microns in diameter.
23. The apparatus of claim I, wherein the capture mechanism of each site comprises
an electric field trap capable of producing a potential energy well for capturing the
single cell.
24. The apparatus of claim 23, wherein the electric field trap comprises electrodes.
20 25. The apparatus of claim 24, further including four electrodes arranged in a
trapezoidal configuration.
26. The apparatus of claim 24, wherein the electric field trap is three-dimensional.
27. The apparatus of claim 24, wherein the electrodes are thin-film poles.
28. The apparatus of claim 26, wherein the electrodes are formed from gold.
25 29. The apparatus of claim 23, wherein the release mechanism includes removing the
potential energy well for ejecting the single cell out of the site.
38
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30. Method of making a cell sorting apparatus, comprising the steps of:
forming a well on one surface of a first substrate, the well being
configured and dimensioned to hold a single cell;
forming a chamber on an opposite surface of the first substrate;
forming a channel in the first substrate to connect the well and chamber
together and permit fluid communication therebetween;
forming a heating element on a second substrate;
positioning the heating element under the chamber; and
attaching the first substrate onto the second substrate such that the second
substrate forms the bottom of the chamber.
31. The method of claim 30, wherein the steps of forming the well, channel and
chamber further comprise etching the first substrate.
32. The method of claim 31, wherein the first substrate comprises a silicon wafer.
33. The method of claim 3 1 , wherein the steps of etching further comprise:
growing thermal oxide onto a first surface of a the silicon wafer substrate;
patterning the oxide using a first mask that defines the shape of the well;
spinning photoresist on top of the oxide;
patterning the oxide using a second mask that defines the shape of the
channel;
etching the wafer to form the channel using the second mask;
etching the wafer to form the well using the first mask;
depositing photoresist on an opposite surface of the silicon wafer
substrate;
patterning the photoresist using a third mask that defines the shape of the
chamber; and
etching the wafer to form the chamber, the chamber having sufficient
depth to connect with the channel.
39
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34. The method of claim 30, wherein the step of forming the heating element
comprises:
spinning photoresist onto the second substrate;
patterning the photoresist with a mask that defines the shape of a heating
element;
selectively removing the photoresist to expose a region of the second
substrate in the shape of the heating element; and
depositing a metallic conductor on the exposed region.
35. The method of claim 34, wherein the step of depositing a metallic conductor
further comprises:
evaporating at least one metal onto the second substrate; and
selectively removing the metal from the substrate.
36. The method of claim 35, wherein the step of selectively removing the metal
further comprises treating the substrate with acetone to remove excess photoresist
and metal deposited on the photoresist.
37. The method of claim 30, wherein the second substrate comprises glass.
38. The method of claim 30, wherein the step of attaching the first substrate onto the
second substrate further comprises joining the first and second substrates together
with an adhesive.
40
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FIG. I A FIG. IB
FIG. IC FIG. ID
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32
32
32
32
34
32
FIG. 3 A
34
^ 9-
^32 32 S
32
FIG. 3B
FIG. 4
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FIG. 6
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FIG. 8
a
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- ; - 300
200-
100-
FIG. IO
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12 mm
< ►
1.5 mm
FIG. IIA
lOOum
FIG. I IB
Cell Well
•* ►
Narrow Channel
FIG. 12 A
FIG. 12 B
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Mask 1
FIG. 13 A
Mask 2
FIG. I3B
Mask 3
FIG. I3C
Mask 4
FIG. 14 A
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O bjQ
CO G
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\h\ BH HB T7V7
O
■ ' vy/i ' Vi ■ i, h">
o
Glass
Slide
Silicon
Chip
PDMS Gasket
FIG. 16 A
Metal
Spacer
PDMS
Plexiglass
FIG. 16 B
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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
17- May 2001 (17^5.200f>
PCT
nun ii in i ii ii ii iii mi in ii
(10) International Publication Number
WO 01/35071 A3
(51) International Patent Classification 7 : COIN 15/14
(21) International Application Number: PCT/USOO/30815
(22) International Filing Date:
1 0 November 2000 ( 1 0. 1 1 .2000)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
60/164.643 10 November 1999 (10.1 1.1999) US
(71) Applicant (for al/ designated States except US): MASS-
ACHUSETTS INSTITUTE OF TECHNOLOGY
I US/US]; 77 Massachusetts Avenue, Cambridge, MA
02131 (US).
(72) Inventors; and
(75) Inventors/Applicants (for US onfy/: BRAFF, Rebecca
I US/US]; 488 Columbus Avenue. #4. Boston. MA 021 18
(US). VOLDMAN, Joel [US/US]: 35 Davis Square.
#6, Somerville, MA 02144 (US). GRAY, Martha, L.
[US/US]: 226 Pleasant Street. Arlington. MA 02476
(US). SCHMIDT, Martin, A. fUS/USl: 78 Ashley Place.
Reading. MA 01867 (US). TONER, Mehmet [US/US|:
100 Pilgrim Road, Wellesley. MA 02481 (US).
(74) Agents: ENGELLENNER, Thomas, J.elal.: Nutter. Me-
Clennen & Fish, LLP. One International Place. Boston. MA
021 10-2699 (US).
(81) Designated States (national): AE, AG. AL, AM, AT. AU.
AZ. BA, BB. BG. BR. BY, CA. CH. CN. CR. CU. CZ. DE,
DK. DM, DZ. EE. ES. FI. GB. GD. GE, GH, GM, HR, HU.
ID, IL. IN. IS, JP, KE. KG. KP. KR. KZ. LC. LK. LR, LS.
LT, LU, LV, MA. MD. MG. MK, MN. MW. MX. MZ, NO,
[Continued on next page]
(54) Title: CELL ANALYSIS AND SORTING APPARATUS FOR MANIPULATION OF CELLS
Resistor
20
10
J
i
Microbubble
22
O
(57) Abstract: A cell analysis
and sorting apparatus is capable of
monitoring over lime the behaviour of
each cell in a large population of cells.
The cell analysis and sorting apparatus
contains individually addressable cell
locations. Each location is capable of
capturing and holding a single cell, and
selectively releasing that cell from that
particular location. In one aspect of
the invention, the cells are captured and
held in wells, and released using vapor
bubbles as a means of cell actuation.
In another aspect of the invention, the
cells are captured, held and released
using electric fields traps.
WO 01/35071 A3 1111111111111111111111111111111111111
NZ. PL. PT. RO. RU. SIX SE. SG. SL SK, SL. TJ. TM. TR. Published:
TT, TZ. UA. UG, US, UZ, VN. YU, ZA. ZW. — with international search report
(84) Designated States (regional): ARIPO patent (GH. GM.
KE. LS, MW, MZ. SD. SL. SZ. TZ. UG. ZW). Eurasian
patent (AM. AZ, BY. KG, KZ. Ml). RU TL TM ). European
patent (AT BE. CM. CY, DE. DK. ES. FL FR. GB. GR. IE,
IT. LU. MC. NL. PT, SE, TR). OAPI patent (BF. BJ. CF.
CG. CI. CM. GA. GN. GW, ML. MR. NE. SN. TIX TG).
(88) Date of publication of the international search report:
21 February 2(K)2
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the I'CT Gazette.
1
INTERNATIONAL SEARCH REPORT
In ational Application No
PCT/US 00/30815
A. CLASSIFICATION OF SUBJECT MATTER
IPC 7 G01N15/14
According lo International Patent Classification (IPC) or to both national classitication and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classitication system followed by classitication symbols)
IPC 7 G01N C12M
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and. where practical, search terms used)
EPO-Internal
C DOCUMENTS CONSIDERED TO BE RELEVANT
Category •
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to daim No.
X
X
A
US 5 506 141 A (DEUTSCH MORDECHAI ET AL)
9 April 1996 (1996-04-09)
column 7, line 37 -column 8, line 25
column 9, line 56 -column 10, line 20
column 11, line 3-22
column 11, line 49 -column 12, line 13
column 14, line 40 -column 16, line 22
column 19, line 24 -column 20, line 42
W0 99 31503 A (SCHMIDT CHRISTIAN ;V0GEL
HORST (CH)) 24 June 1999 (1999-06-24)
page 4, line 15 -page 5, line 30
page 7, line 20-34
page 8, line 15 -page 9, line 31
page 13, line 24-32
_/-
1-6
1-4,6,23
7,30
| )( j Further documents are listed in the continuation of box C. |x [ Patent famlry members are listed In annex.
* SpedaJ categories of died documents : ajm ^ ^ ^ jntema|iona) fiHng ^
. , , . , . . . . . or priority date and not in conf ttct with the application but
•A' document defining the general state of the art which is not dled , 0 understand the principle or theory underlying the
considered to be of parbcular relevance invention
'E* earlier document but published on or after the international . x . docurnent o! particular relevance; the claimed invention
filing date cannot be considered novel or cannot be considered to
■L" document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken alone
which is cited to establish the publication date of another . Y . document o! particular relevance; the claimed invention
[ atatton or other special reason (as specified) cannot ^ considered to involve an inventive step when the
"0* document referring to an oral disclosure, use. exhibition or document is combined with one or more other such docu-
other means ments. such combination being obvious to a person skilled
•P' document published prior to the international filing date but ' n lne art -
later than the priority date claimed document member of the same patent family
Date of the actual completion of the international search
3 August 2001
Dale ot mailing of the international search report
10/08/2001
Name and mailing address of the ISA
European Patent Office. P.B. 5818 Patent laan 2
NL - 2280 HV Rijswtjk
Tel (+31-70) 340-2040. Tx. 31 651 epo nl.
Fax (+31-70) 340-3016
Authorized officer
Zinngrebe, U
Form PCT/1SA/210 (second shoot) (Jury 1092)
page 1 of 2
.a
j
INTERNATIONAL SEARCH REPORT
lr. ational Application No
PCT/US 00/30815
C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT
Category •
Citation ot document, with indication .where appropriate, ot the relevant passages
A
A
DE 197 12 309 A (NMI
NATURWISSENSCHAFTLICHES UN)
20 May 1998 (1998-05-20)
abstract
US 4 894 343 A (TANAKA SHINJI ET AL)
16 January 1990 (1990-01-16)
abstract
1-3
1-3.7
Fo«m PCT/1SA/210 (continumion of second sheet) (July 1992)
page 2 of 2
INTERNATIONAL SEARCH REPORT
Information on patent family members
Ir. ationai Application No
PCT/US 00/30815
Patent document
cited in search report
Publication
date
Patent family
member (s)
Publication
date
US
5310674
A
10-05-1994
AT
29070
T
15-09-1987
AU
_ .562301
B
04-06-1987
AU
1401483
A
17-11-1983
BR
8302395
A
10-01-1984
CA
1202870
A
08-04-1986
CS
8303223
A
12-05-1989
DE
3373143
D
24-09-1987
DK
190583
A
11-11-1983
EP
0094193
A
16-11-1983
ES
522207
D
16-09-1984
ES
8407592
A
16-12-1984
HU
195245
B
28-04-1988
IL
68507
A
31-01-1986
IN
159538
A
23-05-1987
IN
168081
A
02-02-1991
JP
1607661
C
13-06-1991
JP
2034597
B
03-08-1990
JP
59031685
A
20-02-1984
KR
8701670
B
21-09-1987
MX
163377
B
06-05-1992
NO
831637
A,B,
11-11-1983
NZ
204056
A
05-12-1986
SU
1776352
A
15-11-1992
US
4729949
A
08-03-1988
US
5272081
A
21-12-1993
ZA
8303141
A
25-01-1984
AT
71664
T
15-02-1992
AU
594641
B
15-03-1990
AU
3672884
A
03-06-1985
DE
3485462
D
27-02-1992
DK
308385
A
05-09-1985
EP
0162907
A
04-12-1985
JP
61501126
T
12-06-1986
WO
8502201
A
23-05-1985
US 5506141
09-04-1996
WO 9931503
A
24-
■06-
1999
AU
EP
8237998 A
1040349 A
05-07-1999
04-10-2000
DE 19712309
A
20-
•05-
1998
WO
EP
9822819 A
0938674 A
28-05-1998
01-09-1999
US 4894343
A
16-
-01-
1990
JP
JP
2662215 B
63129980 A
08-10-1997
02-06-1988
Foim PCT/lSA/210 (patent lamily annex) (July 1992)
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